Subcellular Mechanisms in Pathogenesis of Pulmonary Arterial Hypertension

描述（由申请人提供）：特发性肺动脉高压（PAH）是一种持续进展的疾病，其特征是内皮细胞和平滑肌细胞增生、增大和空泡化，对细胞凋亡具有抗性。在关于多环芳烃发病机制的讨论中，特别缺少对亚细胞膜和蛋白/受体在质膜和细胞内部之间转运机制的考虑。我们将验证以下假设：在这种疾病中，血管相关蛋白的全部改变（包括各种受体、信号通路分子和eNOS水平的增加/减少）主要是由于高尔基体水平的细胞内运输缺陷导致这些蛋白错误定位到错误的亚细胞区室（“高尔基阻断假说”）。我们认为新参与者（各自的高尔基系带、SNAREs、1-SNAP和NSF）和旧参与者（错误定位的eNOS和减少的亚细胞NO，突变的BMPRII物种）在亚细胞机制中“导致”PAH。目的1将处理高分辨率3d成像研究，在不同的ph模拟条件下[NO清除，缺氧和单根草碱（MCTP）]，在原代HPAECs和HPASMCs培养中高尔基体形态，扩大和碎裂的变化，重点是巨肽，GM130, p115， GS28和1-SNAP。Aim II将从eNOS亚细胞错误定位的角度处理这些变化的后果。这些研究将在(1)暴露于no清除、缺氧或MCTP的HPAECs培养物中进行，以及(2)来自mct治疗的大鼠、人PAH和感染shiv -nef的肺血管病变猕猴的肺血管病变福尔马林固定部分的细胞中进行。Aim III将验证pah病相关的BMPRII突变种在HPAECs和HPSMCs中错定位eNOS和运输中介蛋白的假设，从而对细胞内运输产生显性负作用。综上所述，提出的研究代表了在PAH中测试高尔基阻断假说的下一步。在该项目完成后，我们将在高分辨率3d细胞培养和组织细胞中阐明该病高尔基体形态的变化，获得eNOS和1-SNAP亚细胞错定位的证据，并提供BMPRII突变如何破坏细胞内运输的新机制，最终导致PAH病变肺血管细胞中观察到的细胞表型。

DESCRIPTION (provided by applicant): Idiopathic pulmonary arterial hypertension (PAH) is an unrelentingly progressive disease characterized by proliferative, enlarged, and vacuolated endothelial and smooth muscle cells resistant to apoptosis. Singularly missing from discussions of the pathogenesis of PAH is consideration of subcellular membrane- and protein/receptor-trafficking mechanisms between the plasma membrane and the cell interior. We will test the hypothesis that the entire basket of alterations in vasorelevant proteins (including increases/decreases in levels of various receptors, signaling pathway molecules and eNOS) in this disease is largely due to mislocalization of these proteins to the wrong subcellular compartments due to defects in intracellular trafficking at the level of the Golgi apparatus ("Golgi blockade hypothesis"). We implicate both new players (respective Golgi tethers, SNAREs and 1-SNAP and NSF) and old players (mislocalized eNOS and reduced subcellular NO, mutant BMPRII species) in the subcellular mechanisms that "cause" PAH. Aim I will deal with high-resolution 3-D imaging studies of the alterations of the Golgi apparatus morphology, enlargement and fragmentation under various PAH-mimetic conditions [NO scavenging, hypoxia and monocrotaline pyrolle (MCTP)] in cultures of primary HPAECs and HPASMCs with a focus on giantin, GM130, p115, GS28 and 1-SNAP. Aim II will deal with the consequences of these changes in terms of the subcellular mislocalization of eNOS. The studies will be carried out in (1) cultures of HPAECs exposed to NO-scavenging, hypoxia or MCTP, and (2) in cells in formalin-fixed sections of vascular lesions in lungs derived from the MCT-treated rat, human PAH and the SHIV-nef-infected macaque with pulmonary vasculopathies. Aim III will test the hypothesis that PAH-disease-associated BMPRII mutant species can mislocalize eNOS and trafficking-mediator proteins in HPAECs and HPSMCs causing a dominant-negative effect on intracellular trafficking. Taken together, the proposed studies represent the next step in testing the Golgi blockade hypothesis in PAH. Upon completion of this project we will have elucidated changes in Golgi apparatus morphology in high-resolution 3-D in both cell culture and in cells in tissue sections in this disease, obtained evidence for subcellular mislocalization of eNOS and 1-SNAP, and provide a novel mechanism for how mutations in BMPRII might disrupt intracellular trafficking culminating in the cellular phenotype observed in pulmonary vascular cells in PAH lesions. PUBLIC HEALTH RELEVANCE: Fifteen thousand patients die of pulmonary arterial hypertension (PAH) every year in the U.S. It is not clearly understood what causes PAH. Although mutations in the BMPR2 gene have been implicated, the mechanisms have not been elucidated despite 10 years' of investigations. We propose a novel way of looking at the pathogenesis of this disease in terms of a global disruption of intracellular trafficking in pulmonary arterial endothelial and smooth muscle cells which result in the incorrect placement of vasorelevant proteins on the cell surface and in locations within the cells.